Cover Page. The handle holds various files of this Leiden University dissertation.

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1 Cover Page The handle holds various files of this Leiden University dissertation. Author: Flinterman, Linda Elisabeth Title: Risk factors for a first and recurrent venous thrombosis Issue Date:

2 Risk factors for a first and recurrent venous thrombosis Linda Flinterman

3 Risk factors for a first and recurrent venous thrombosis

4 ISBN: Layout and Printing: Off Page, Cover: Rob Severein iamartdirector.com Copyright 2013 by L.E. Flinterman. All rights reserved. No part of this book may be reproduced, stored in a retrieval system, or transmitted in any form or by any means, without prior permission of the author.

5 Risk factors for a first and recurrent venous thrombosis PROEFSCHRIFT ter verkrijging van de graad van Doctor aan de Universiteit Leiden op gezag van de Rector Magnificus prof.mr. C.J.J.M. Stolker, volgens besluit van het College van Promoties te verdedigen op dinsdag 28 mei 2013 klokke 15:00 uur door Linda Elisabeth Flinterman geboren te Delft in 1984

6 Promotie commissie Promotor: Co-promotores: Overige Leden: Prof. Dr. F.R. Rosendaal Dr. S.C. Cannegieter Dr. A. van Hylckama Vlieg Prof. Dr. K. Overvad (Universiteit van Aarhus, Denemarken) Prof. Dr. S. Middeldorp (Universiteit van Amsterdam) Dr. S. le Cessie The work described in this thesis was performed at the department of Clinical Epidemiology of het Leiden University Medical Center in Leiden, the Netherlands. The research described in this thesis was supported by a grant of the Dutch Heart Foundation (DHF-2008B086). Financial support by the Dutch Heart Foundation for the publication of this thesis is gratefully acknowledged.

7 Contents Chapter 1 General Introduction 7 Chapter 2 Chapter 3 Chapter 4 Chapter 5 Chapter 6 Chapter 7 Chapter 8 Current Perspective of venous thrombosis of the upper extremity 15 Venous thrombosis of the upper extremity: effect of blood group and coagulation factor levels on risk 23 Recurrent thrombosis and survival after a first venous thrombosis of the upper extremity 35 Long-term survival in a large cohort of patients with venous thrombosis: incidence and predictors. 49 Chronic obstructive pulmonary disease and pulmonary e mbolism 63 Incidence and characteristics of recurrent venous thrombosis in a large cohort of patients with a first venous thrombosis 77 Body height, mobility, and risk of first and recurrent venous thrombosis 93 Chapter 9 Summary and General Discussion 105 Chapter 10 References 119 Nederlandse samenvatting 127 Dankwoord 129 Curriculum Vitae 131

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9 1 General Introduction

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11 Venous thrombosis When a blood vessel is damaged it is important that bleeding stops rapidly. In healthy individuals the coagulation system is triggered immediately. A clot is formed to stop the bleeding and to enable vessel repair. After the vessel wall integrity is re-established the clot is dissolved by the fibrinolytic system. Venous thrombosis is a result of a disturbance of the balance of the pro- and anticoagulant system or the pro- and antifibrinolytic system towards the prothrombotic side. This disturbance can be caused by acquired and genetic risk factors. In the 19th century Virchow proposed a theory in which he considers three causes of thrombosis, i.e. alterations of the blood flow (stasis), vascular endothelial injury, or alterations in the constitution of the blood. These three conditions are known as Virchow s triad, which is still valid today 1. 1 General introduction Venous thrombosis is an occlusion by a blood clot of one or more of the veins; this occurs mostly in the leg but may affect every vein in the body. The occlusion of a vein in the leg may cause swelling, redness and pain of the affected limb. When part of a clot dislodges it can travel up through the lower caval vein through the heart to the lungs were occlusion of pulmonary vessels leads to symptoms of pulmonary embolism. Venous thrombosis occurs in 1-2 per 1000 persons per year 2. The incidence increases with age from 1 per in the young up to 1 per 100 in the elderly 3. About one third of the thrombotic clots form emboli that cause pulmonary emboli 2,3. Complications of venous thrombosis include pulmonary hypertension (incidence 3% per year) 4, the post thrombotic syndrome (cumulative incidence 25-50% after 1-2 years) 5, and bleeding due to treatment with anticoagulants (incidence of major bleeding % per year) 6. Venous thrombosis is a potentially lethal disease due to pulmonary embolism. Pulmonary emboli are lethal in about 20% of the cases 3. Mortality remains elevated for several years after the thrombotic event, mainly for patients with a first idiopathic venous thrombosis or patients with venous thrombosis due to malignancy 3. Twelve percent of patients with venous thrombosis without malignancy die within one year after the thrombotic event. This implies that venous thrombosis has a major impact on mortality 3,7. Risk factors for a first venous thrombosis Venous thrombosis is a multicausal disease. Many genetic and acquired risk factors have been described that increase the risk of venous thrombosis. Furthermore, many of these risk factors interact with each other, resulting in a higher risk of a thrombotic event than expected based on the separate effects of these factors 8,9. Most genetic risk factors known are variations in genes that are involved in the coagulation system, i.e., mutations in protein C and S genes, factor V Leiden (FVL, rs6025) and the prothrombin G20210A mutation (rs ), FGG and Von Willebrand Factor (VWF) 10,11. In a clinical setting screening for FVL 12 and PT20210A 13 after a venous thrombotic event is often performed, 9

12 1 General introduction although the clinical utility is disputed. The FVL mutation is prevalent with about 5% of the Caucasian population carrying this mutation. FVL increases the risk of venous thrombosis 5-fold and interacts synergistically with several acquired risk factors, e.g, oral contraceptive use and pregnancy. The factor V Leiden mutation reduces the ability of protein C to inactivate factor V. This reduced deactivation of factor V results in a prolonged activation of the coagulation system. The prothrombin mutation is less common than FVL, about 2% of the Caucasian population carries this mutation 13. The PT20210A mutation is associated with elevated levels of prothrombin. PT20210A increases the risk of venous thrombosis about 3-fold 13. Several other single nucleotide polymorphisms (SNPs) have been described to mildly increase the risk of venous thrombosis with relative risks varying from 1.2 to Known acquired risk factors are increasing age, oral contraceptive use, pregnancy, surgery, minor injuries, trauma, hormone replacement therapy, long haul flights, malignancies, and immobilization 7, They are either associated with stasis or a prothrombotic state by affecting the coagulation system. Most of these factors are known to interact with genetic risk factors increasing the risk of venous thrombosis even further 19,20. Despite this extensive knowledge on risk factors, the cause of venous thrombosis remains unknown in about one third of patients. Recurrent venous thrombosis After a first venous thrombosis 15-40% of the patients will experience one or more recurrent events within 5 years 3, Treatment with anticoagulation therapy after a venous thrombosis stops the clot from growing but does not dissolve it. The period in which the clot will be completely dissolved, if ever, depends on the size of the clot and differs per patient. Therefore, when a patient develops new symptoms after discontinuation of treatment for venous thrombosis it is often difficult to distinguish between old and new thrombi. In the literature, varying definitions of recurrence are used. Some studies perform regular ultrasounds to follow the dissolving of the clot, or growth of an old clot as proof of a recurrence 25,26. Some studies define a recurrence as a thrombus found in patients who have new complaints after the first event, while others provide no definition at all 27,28. However, most studies agree on defining as a recurrent event a thrombus in a new vein segment compared to the first event found on ultrasound 22, This last definition might be incorrect when a first event was a thrombus of the leg and recurs as a pulmonary embolism. In theory the partly dissolved clot, which caused the first event, could be dislodged and causing the pulmonary embolism. While this would then need to be classified as the first event, it may easily be regarded as a recurrence. Due to these difficulties in diagnosis and differences in definition of recurrence the incidence of recurrent venous thrombosis varies widely in the literature. Risk factors for a recurrent event seem to be different from those for a first event. Some of these differences can easily be explained because risk factors were of a transient nature or because prophylactic treatment is given whenever these factors reoccur (surgery, minor 10

13 injuries and pregnancy 22 ). However, age, which is one of the major risk factors does not seem to increase the risk of recurrence, nor do genetic prothrombotic abnormalities The main risk factors for recurrence, which are currently known, are male sex and an idiopathic first event. Sex is not a major risk factor for first venous thrombosis. For a first venous thrombosis the risk is higher in women up to the age of 40 than in men, after the age of 40 the risk is higher in men than in women. Although it may seem that the difference in recurrence risk can easily be explained by discontinuation of oral contraceptives and prophylaxis during subsequent pregnancies, previous studies showed that this does not explain the difference in risk between men and women for a recurrent event 36,40. Venous thrombosis of the upper extremity Venous thrombosis of the upper extremity is a rare form of venous thrombosis that is present in 4% of all patients with venous thrombosis 41. Risk factors for this form of thrombosis are mainly similar to those for venous thrombosis of the leg. However, venous thrombosis of the upper extremity is most frequently caused by either a central venous catheter or the Paget-Schrötter syndrome 42 44, which factors are specific for the upper extremity. 1 General introduction Study populations MEGA study The Multiple Environmental and Genetic Assessment study of risk factors for venous thrombosis (MEGA study) is a large case-control study with over 5000 cases and over 5000 controls. Consecutive patients aged between (n=5170) with a first venous thrombosis of the leg, arm, or pulmonary embolism were collected at six anticoagulation clinics in the Netherlands (Amersfoort, Amsterdam, Leiden, Rotterdam, The Hague, and Utrecht) between March 1999 and September Control subjects were either partners of the patients (n=3297) or were collected via random digit dialing (RDD) (n=3000), RDD controls came from the same area as the anticoagulation clinics and were frequency matched by age and sex to the cases. Patients and controls were asked to fill in an extensive questionnaire about their medical history and potential risk factors for venous thrombosis. Until June 2002, all participants were asked to provide a blood sample. From June 2002 onwards only DNA samples were taken with buccal swabs. Blood samples of patients were drawn at least 3 months after discontinuation of anticoagulation treatment or, when treatment was continued for more than one year, approximately one year after the thrombotic event. Partners of the patients provided blood samples at the same time as the case. Controls from the random digit dial group provided blood samples within a few weeks after the questionnaire was sent. MEGA follow-up study The MEGA follow-up study is a follow-up of the participants of the MEGA study described above. Survival status of all participants was checked at the community registries in

14 1 General introduction Additionally, all cases and controls were followed in time with a median follow-up of five years. All patients and controls that indicated they were willing to participate in a follow-up study at the initial MEGA study were sent follow-up questionnaires between June 2008 and June These questionnaires comprised, for all participants, questions about the occurrence of arterial thrombosis and potential risk factors for both arterial and venous thrombosis. For the cases additional questions were added about the post-thrombotic syndrome and recurrent venous thrombosis. Recurrent events were adjudicated with information from discharge letters and anticoagulation clinics. The aim of the MEGA follow-up study was to assess the incidence of recurrent venous thrombosis, to identify new risk factors for a recurrent venous thrombosis, and to study the association between venous and arterial thrombosis and their common risk factors. Outline thesis This thesis focuses on risk factors for a first and recurrent venous thrombosis of both the lower and upper extremity. Chapter 2, 3 and 4 concern the risk of thrombosis of the upper extremity and the risk factors for recurrence in these patients. In chapter 2 the current state of knowledge regarding risk factors for venous thrombosis of the upper extremity is reviewed. Blood type non-o and increased levels of FVIII, FXI and FIX are known to increase the risk of a first venous thrombosis of the leg. However, this had not been studied for venous thrombosis of the arm. In chapter 3 we investigate the influence of blood group and levels of coagulation factors on the risk of a first venous thrombosis of the upper extremity. In chapter 4 risk factors for a recurrent venous thrombosis after a first venous thrombosis of the upper extremity are studied. About 15% of the patients die shortly after a venous thrombosis event. However, the long-term effect on mortality in these patients was not well known. Chapter 5, 6, 7 and 8 concern venous thrombosis of the leg. Both risk factors for a first and recurrent event, and the survival after a first venous thrombosis were studied. In chapter 5 we describe the long-term mortality after a first venous thrombosis. From chapter 5 we learned that patients with a first venous thrombosis had a 3.5-fold increased risk to die of chronic obstructive pulmonary disease (COPD). In chapter 6 we studied COPD as a risk factor for a first venous thrombosis. The incidence and definition of recurrent venous thrombosis varies widely in the literature. In chapter 7 we describe the incidence of a recurrent event in detail as well as the influence of sex, age and an idiopathic first venous thrombosis on recurrence. In chapter 8 we examine body height as a risk factor for both first and recurrent venous thrombosis for both men and women; until now this association was mainly studied in men for a first venous thrombosis. 12

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17 2 Current Perspective of venous thrombosis of the upper extremity Journal of Thrombosis and Haemostasis 2008;6: L.E. Flinterman F.J.M van der Meer F.R. Rosendaal C.J.M. Doggen

18 2 Current perspective of venous thrombosis of the upper extremity Summary Venous thrombosis of the upper extremity is a rare disease. Therefore, not as much is known about risk factors, treatment and the risk of recurrence as for venous thrombosis of the leg. Only central venous catheters and strenuous exercise are commonly known risk factors for an upper extremity venous thrombosis. In this review an overview of the different risk factors, possible treatments and the complications for patients with a venous thrombosis of the upper extremity is given. 16

19 Introduction Venous thrombosis of the upper extremity (UEDVT), defined as a thrombus in the subclavian, axillary or brachial vein, accounts for 4-10% of all venous thromboses This figure may be an underestimation given that UEDVT is often asymptomatic 44,51,52. The low incidence of UEDVT compared with the leg may be explained by the lower gravitational stress. Additionally, the veins of the upper extremities contain fewer valves than the legs which could be potential foci for thrombus formation. Furthermore, stasis contributing to the occurrence of thrombosis is very rare in the upper extremity. The most likely conditions for stasis to occur are surgery and plaster cast of the upper extremity 41,52. Primary venous thrombosis UEDVT is usually divided into primary and secondary thrombosis 48,53. Primary UEDVT includes idiopathic thrombosis and thrombosis associated with the thoracic outlet syndrome or effort (Paget-Schrötter syndrome) 42,43. The incidence of primary UEDVT is 2 per person-years 46,54 and accounts for approximately 30% of all UEDVT 48,54,55. Thoracic outlet syndrome (TOS) relates to various forms of compression in the thoracic outlet. The thoracic outlet is located between the base of the neck and the axilla. Compression of the thoracic outlet can either be on the brachial plexus or on the blood vessels in the outlet. Compression of both nerves and vessels is rare. In approximately 3-10% of the cases, TOS will be vascular which can be divided into venous or arterial TOS 56. UEDVT can be caused by venous TOS. In venous TOS the thoracic outlet veins are compressed by the clavicle and the first rib 57. This will result in compression or sudden occlusion of the vein. Approximately 60% of patients with primary UEDVT have the thoracic outlet syndrome 47. Treatment consists of either anticoagulation alone or in combination with resection of the first rib 57,58. Paget-Schrötter syndrome, also called effort thrombosis, is a manifestation of TOS. The syndrome mostly occurs in young healthy persons, related to strenuous activity of the arm during sports The effort causes microtrauma of the vessel intima by repeated compression of the clavicle and first rib, which activates the coagulation 46. The compression is mostly caused by trauma or by the enlarged muscles in the shoulder girdle 48,56,58,60,64,66. 2 Current perspective of venous thrombosis of the upper extremity Secondary venous thrombosis Secondary UEDVT is thrombosis with a known risk factor. These risk factors can be genetic or acquired 53,67,68. Central venous catheters The main risk factor for UEDVT is a central venous catheter(cvc) 49,67. The catheters are used to provide chemotherapy, medication and parenteral nutrition and for administration of fluids, blood products and hemodialysis 44. Several studies investigated 17

20 2 Current perspective of venous thrombosis of the upper extremity the incidence and risk factors for UEDVT in patients with a CVC, as well as prophylaxis to prevent thrombosis CVCs can be divided into peripherally inserted catheters, chest catheters and pacemakers. Peripherally inserted catheters have an incidence of thrombosis from 10% to 38% 69,70,77. The incidence of UEDVT for chest catheters ranges from 2% to 41% 71,72,75,77. The risk of thrombosis is dependent of the diameter of the catheter; a larger diameter gives a higher risk of thrombosis 70. The risk of UEDVT is lower for polyurethane and silicone catheters compared with polyethylene or Teflon-coated catheters 78,79. Pacemakers give thrombosis in 10%. This risk increases with the number of leads from the pacemaker 80,81. Most occlusions occur within the first 2 months after insertion of the catheter 70,82,83. Malignancy Malignancy is an important risk factor for UEDVT. However, the increased risk in patients with malignancy is mainly induced by CVCs Dhami & Bona, 1993; Girolami, Prandoni, Zanon, Bagatella, & Girolami, 1999) 86. In the Multiple Environmental and Genetic Assessment (MEGA) study of risk factors for venous thrombosis, a population-based case-control study, 179 patients with UEDVT were compared to 2399 control subjects. In patients without a CVC with an active form of malignancy an 18-fold increased risk of UEDVT compared with patients without active malignancy was found. Active malignancy was defined as malignancy diagnosed 5 years or less before first venous thrombosis 86. A 7.7-fold increased risk of UEDVT was found when all, active and inactive, malignancies were included. Coagulation abnormalities and genetic risk factors In the literature the prevalence of coagulation abnormalities in patients with UEDVT ranges from 8% to 61% 45,68,87,88. The differences can be explained by the size of these studies, including only between 18 and 51 patients. Additionally, the studies had different time points of blood collection: at diagnosis of thrombosis, 3 weeks after the event or after discontinuation of anticoagulation therapy. This can influence the assessment of coagulation abnormalities. Only antiphospolipid antibodies seem consistently more frequently present compared to the healthy population Factor (F) V Leiden 12 and the prothrombin 20210AP 13 mutation are common genetic risk factors in venous thrombosis of the leg, and may be a risk factor for UEDVT. Some studies reported an increased risk for either factor V Leiden or the prothrombin 20210A mutation or both. An Italian study included 115 patients with an UEDVT and 797 control subjects. A 5- to 6-fold increased risk of UEDVT was found for patients with one of the mutations 90. In a Spanish study of 79 cases and 165 controls an increased risk was found for patients with the prothrombin 20210A mutation, no effect was found for the FV Leiden mutation 53. A follow-up study including 257 patients with a CVC found a 3-fold increased risk of a UEDVT in patients with a mutation compared with patients without a mutation 74. Although the majority of the other studies showed an increased risk for patients with the 18

21 FV Leiden or prothrombin 20210A mutation, the number of patients in these studies was small. Therefore, no solid conclusions can be drawn. Oral contraceptive use, surgery and plaster cast Oral contraceptive use did not increase the risk of UEDVT in most studies 48,87,90,91. However, in a Spanish study a 5.7-fold (CI: ) increased risk was found, and the MEGA study found a 2-fold (CI: ) increased risk 53,86. So, there is no consensus about oral contraceptive use as risk factor for UEDVT. Differences may be explained by the type of patients included in the studies. A study including patients with a primary UEDVT showed an increased risk while studies including patients with secondary UEDVT did not show an effect of oral contraceptives. Surgery and plaster cast of the upper extremity result in stasis in the upper extremity. Only one study investigated surgery and plaster cast as a risk factor of UEDVT. This study found surgery of the upper extremity as a risk factor with an odds ratio of 13.1 (95% CI ) 86. Plaster cast of the arm increased the risk with an odds ratio of 7.0 (95% CI ) 86. Other risk factors Other possible risk factors for UEDVT are obesity, hormone replacement therapy (HRT) and pregnancy. Few studies have investigated these risk factors. Obesity and HRT were no risk factors 86,92. A case series reported pregnancy, especially in combination with ovary hyperstimulation syndrome, as a risk factor in women for UEDVT 93. However no large studies were performed to assess the thrombotic risk for pregnancy. Treatment Unfortunately, no randomised controlled trials have been performed on the optimal treatment for patients with UEDVT. Therefore, there is still debate about what the best therapy is and how side effects can be minimized. Anticoagulation is the most common treatment with a similar strategy as for deep vein thrombosis of the leg consisting of low molecular weight heparin and vitamin K antagonists. Removal of the main risk factor, for instance a CVC, probably reduces the risk of a recurrent event. However, in most cases removal of the CVC is not possible and therefore is inserted in the other arm or on the opposite site of the chest. Thrombolysis, surgery, thrombectomy and balloon dilatation with and without stent placing are used far less In these cases removal of a rib or correction of malformations of the ribs or clavicle may be part of the treatment. However, these aggressive forms of treatment may have serious side effects. Thrombolysis gives a higher risk of bleeding and surgery can cause pneumothorax, nerve damage and rethrombosis 98,99. Balloon thrombectomy and venous stents can cause thrombosis by inducing intima damage 97. In several pilot studies thrombolysis was compared with standard anticoagulation therapy in patients with a UEDVT. Most patients reacted well to thrombolysis and there were only a few more bleedings compared to the standard treatment A clear advantage of thrombolytic therapy has not been shown. Recently, the outcomes of the RIETE registry of patients with 2 Current perspective of venous thrombosis of the upper extremity 19

22 2 Current perspective of venous thrombosis of the upper extremity venous thrombosis showed no difference in outcome after 3 months of therapy in patients with a deep vein thrombosis of the upper extremity or of the leg 103. However, in this study treatment for UEDVT was different from that for thrombosis of the leg. Approximately 50% of the patients with UEDVT were only treated with low molecular weight heparins for 3 months and the other half with vitamin K antagonists. In venous thrombosis of the leg 70% of patients were treated with vitamin K antagonists. Therefore, the groups in this registry were not entirely comparable. These results show that patients with UEDVT with their current treatment have the same prognosis after three months as patients with venous thrombosis of the leg. Whether this is the most optimal treatment remains unclear. Complications The main complications of UEDVT are pulmonary emboli (30%), post-thrombotic syndrome (PTS) and death 55,87, PTS occurs in 7-44% of patients 55,107,108 (table 1). These percentages are based on small studies with different definitions of PTS. Mortality ranges from 15 to 50%, and is high because of major co-morbidity (e.g., malignancy, infection and multi-organ failure) 48,106,108. In a few small studies the annual recurrence rate after a first UEDVT was 2% to 8%. However, groups were small and only one study found thrombophilia as possible risk factor for a recurrence; the other studies were too small to identify risk factors 90,106,108. Table 1. Overview of different studies regarding complications in patients with venous thrombosis of the upper extremity Author N of cases %men Mean age (range) Primary vs Secondary PTS Recurrence (annual) Mortality Hingorani (9-101) Both 7% - 34% Prandoni (19-79) Both 15% 7% 15% Martinelli (14-61) Primary - 2% - Baarslag (23-86) Both 18% 8% 50% Prandoni Both 20% 2% 20% Hingorani (1-101) Both % Kahn (22-86) Both 44% - - Hingorani (9-101) Both % Conclusion UEDVT is a rare disease that mainly occurs in patients with a CVC. Most other risk factors are the same as for patients with a venous thrombosis of the leg. Mortality, pulmonary embolism and PTS are the most important complications of UEDVT but the incidences of these complications vary between studies. The optimal treatment of UEDVT remains unclear. 20

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25 3 Venous thrombosis of the upper extremity: effect of blood group and coagulation factor levels on risk British Journal of Haematology;149: L.E. Flinterman A. van Hylckama Vlieg F.R. Rosendaal C.J.M. Doggen

26 3 UEDVT: effect of blood group and coagulation factor levels Summary Venous thrombosis of the upper extremity is a rare form of thrombosis, accounting for around 4 percent of all venous thromboses, of which only a few risk factors are known. The aim of this case-control study was to investigate the effect of coagulation factors on risk of venous thrombosis of the upper extremity. Patients with venous thrombosis of the arm and partner controls were selected from the MEGA study, a large populationbased case-control study. Participants with a malignancy were excluded. Odds ratios were estimated for elevated levels of factor II, VII, VIII, IX, X, XI, von Willebrand Factor (vwf), and fibrinogen, low levels of protein C, protein S, and antithrombin, and for blood group non-o. Substantially increased risks of venous thrombosis of the upper extremity were found for patients with high levels (above 90 th percentile vs below) of factor VIII (Odds ratio (OR): 4.2, 95% confidence interval (CI): ), vwf (OR: 4.0, 95% CI: ), fibrinogen (OR: 2.9, 95% CI, ), and for blood group non-o compared to O (OR: 2.1, 95% CI, ). The other factors were not associated with an increased risk. Elevated levels of several procoagulant factors are associated with a strongly increased risk of venous thrombosis of the upper extremity. 24

27 Introduction Only 4 % of all cases of venous thrombosis are located in the upper extremity 41,51</sup>. Therefore, most studies on risk factors for venous thrombosis focused on venous thrombosis of the leg or pulmonary embolism and only a few studies investigated the risk of venous thrombosis of the upper extremity, often restricted to a small number of risk factors. Malignancy, a central venous catheter, oral contraceptive use, surgery of the upper extremity, physical activity of the arm and the factor V Leiden 12 and prothrombin 20210A 13 mutation are known to increase the risk of venous thrombosis of the upper extremity in several studies, including the MEGA study44,53,63,72,77,85,86,90. Individuals with elevated levels of procoagulant factors II, VIII, IX, and XI have an increased risk of venous thrombosis of the leg, with two- to threefold increased risks for those in the highest 10 percent of the population distribution 13, Furthermore, blood group non-o is associated with a two-fold increased risk of venous thrombosis of the leg 113. Only a few studies investigated the effect of procoagulant factors in patients with venous thrombosis of the upper extremity. Two studies examined the effect of factor VIII and fibrinogen. High levels of factor VIII were found to be present in the same number of patients with venous thrombosis of the upper extremity as in patients with venous thrombosis of the leg 114. Similarly a high prevalence of high levels of fibrinogen was found 115. However, in both studies no comparisons with the general population were made and hence no risk estimates were reported. Deficiencies of anticoagulants protein C, protein S, and antithrombin substantially increase the risk of venous thrombosis of the leg 116. Studies on the association of these defects with upper extremity thrombosis are scarce and have yielded conflicting results 45,88,91,94. The aim of this study was to investigate the levels of pro- and anticoagulant factors and blood group as risk factors for venous thrombosis of the upper extremity. 3 UEDVT: effect of blood group and coagulation factor levels Methods Study population Analyses were performed as part of a large population-based case-control study. Patients and control subjects were selected from the Multiple Environmental and Genetic Assessment (MEGA) study of risk factors for venous thrombosis. Details of this study have been described elsewhere 7. In the overall study over 5000 consecutive patients with deep venous thrombosis of the leg, a pulmonary embolism, or a venous thrombosis of the upper extremity between the age of 18 and 70, and their partners (>3000) were included. Patients were treated in 6 anticoagulation clinics in the Netherlands. From March 1999 until September 2004, 329 consecutive patients with a venous thrombosis of the upper extremity were invited. Of these 329 patients, 224 participated (68%). There 25

28 3 UEDVT: effect of blood group and coagulation factor levels were no differences in sex and age between those who did and did not participate. Blood was obtained from patients and control subjects included up to July Since the risk factors in the current study involve plasma factor levels, a blood sample is necessary. Consequently, 107 patients and 1825 controls were eligible for this study. Venous thrombosis was objectively confirmed with either ultrasonography, duplex sonography or computed tomography. Partners of patients with venous thrombosis in the overall study were included as control subjects. These were therefore partners of patients with different types of venous thrombosis, including deep-vein thrombosis of the leg and pulmonary embolism. Control subjects did not have a history of venous thrombosis and were between 18 and 70 years. Individuals with a malignancy (31 cases and 57 controls) and individuals without a blood sample (7 cases and 329 controls) were excluded from the present study. In total 69 patients with a venous thrombosis of the upper extremity and 1439 control subjects were included. Data collection All participants were asked to fill in a questionnaire on acquired risk factors for venous thrombosis within a few weeks after the index date. For patients the index date was date of the thrombotic event. For control subjects the index date was the date of diagnosis of thrombosis of their partner. Oral contraceptive use was considered to be present when used within 3 months before the index date. Idiopathic venous thrombosis was defined as present when occurring in the absence of factor V Leiden, prothrombin 20210A mutation, oral contraceptive use, surgery, plaster cast and, injury within 3 months before thrombosis, central venous catheter within a month before thrombosis and pregnancy or puerperium (< 5 weeks after delivery) at time of thrombosis. Both patients and control subjects were invited for a blood draw and interview at least 3 months after discontinuation of vitamin K antagonist treatment of the patient. Infection was present when patients reported a febrile disorder at time of blood draw. When duration of treatment was more than one year patients were invited for a blood draw at least 12 months after the thrombotic event. The mean time between index date and blood draw was 10 months for cases (range 4-24 months) and 7 months for controls (range 1-34 months). All participants filled in an informed consent form and gave permission to obtain information about their medical history. This study was approved by the Ethics Committee of the Leiden University Medical Center, the Netherlands. Laboratory measurements Blood samples were drawn into vacuum tubes containing 0.1-volume mol/L trisodium citrate as anticoagulant. Activity of factor II, VII, VIII, X, and XI was measured with a mechanical clot detection method on a STA-R coagulation analyzer. All measurements were performed following the instructions of the manufacturer (Diagnostica Stago, Asnières, France). Levels of factor IX antigen were determined by enzyme-linked immunosorbent essay (ELISA). Von Willebrand factor antigen was measured with the immuno-turbidimetric method, using the 26

29 STA liatest kit (rabbit anti human VWF antibodies). Fibrinogen activity was measured on the STA-R analyzer according to the method of Clauss. The 20146G/- (rs ), 21463C/G (rs ), 21867A/G (rs ), and 21996C/- (rs ) blood group polymorphisms were determined by a 5 nuclease assay (Taqman; Applied Biosystems, Foster City, California) using a standard PCR reaction mix (Eurogentec, Seraing, Belgium) and an allele specific fluorescent probe equipped with a minor groove binding moiety (Applied Biosystems). Definitions of low levels of antithrombin, protein S, and protein C have been described elsewhere 117. In brief, protein C, protein S, or antithrombin deficiency was defined as value below the mean minus two standard deviations in the control subjects. Due to unreliable test results, individuals with a blood sample at time of pregnancy were excluded from the analysis of protein C and protein S. The same applied to individuals using oral contraceptives at time of the blood sample in the determination of protein S. Sixty-six out of 69 patients and 1430 out of 1439 controls are left for the analysis of protein C and 57 patients vs controls for protein S. The risk of vitamin K dependent factors was determined for 57 patients with a blood draw after discontinuation of vitamin K antagonist treatment. There were no missing data for the pro- and anticoagulant factors. Statistical analysis Odds Ratios (OR) and 95% confidence intervals were calculated as an estimate of the risk of venous thrombosis associated with coagulation factor levels and blood group non-o. Odds Ratios were adjusted for age and sex with a logistic regression model. The 90 th percentile in the control subjects was used as cut off point for factors II, VII, VIII, IX, X, and XI, von Willebrand factor (VWF), and fibrinogen. For those factors with an increased risk for patients above the 90 th percentile, tertiles were defined on the basis of the distribution among control subjects to investigate a dose response relation using the lowest tertile as a reference. Individuals with blood group non-o were compared with individuals with blood group O. The thrombotic risks associated with elevated factor VIII and VWF levels, and blood group were mutually adjusted. Since previous research found a joint effect of factor VIII and oral contraceptive use in patients with a venous thrombosis of the leg, the joint effect of factor VIII above the 90 th percentile with oral contraceptive use among women was assessed Participants with low factor VIII and without oral contraceptive use at the time of index date were used as a reference group. The joint effect was adjusted for age. All analyses were performed using SPSS 14 (SPSS Inc, Chicago, Ill). 3 UEDVT: effect of blood group and coagulation factor levels Results There were slightly more men in the control group than in the patient group: 49 versus 44 percent. Median age of the 69 patients was 39 (5 th -95 th percentile years) compared with 51 (5 th -95 th percentile years) in the 1439 controls (table 1). The prevalence of infection was similar between cases and control subject. White blood cell count was 5.6 versus 5.7 respectively. 27

30 3 Mean levels of procoagulant factors in patients with venous thrombosis of the upper extremity and control subjects are shown in table 2. Mean levels of factor VIII, VWF and fibrinogen were higher among the cases. All other procoagulant factors were similar in cases and controls or even higher in the control than the case group. The distribution of blood group differed for cases and controls. After dichotomization by the 90 th percentile, elevated levels of factors II, VII, IX, and X were not associated with an increased risk of an upper extremity venous thrombosis (table 3). Mean levels of factor VIII, VWF, and fibrinogen were higher in patients than in controls (mean difference: factor VIII (21.7IU/ml, 95%CI, UEDVT: effect of blood group and coagulation factor levels Table 1. Baseline characteristics of the study population, including 69 patients with a venous thrombosis of the upper extremity and 1439 control subjects. Patients N (%) Controls N (%) Sex (% men) 30 (44) 706 (49) Median age (years) CVC* 4 (6) 0 (0) Infection % 19 (27) 321 (23) WBC* (mean, SD) 5.6 (1.4) 5.7 (1.7) Idiopathic thrombosis 22 (32) - Oral contraceptive use # 24 (62) 131 (18) *CVC=Central venous catheter, WBC=White blood cell count # In women (24 out of 39 cases, 131 out of 733 control subjects) at time of the index date % At time of blood sample Table 2. Mean levels of procoagulant factors and the prevalence of blood group in 69 patients and 1439 control subjects Mean level coagulation factor Patients Mean (SD)* Controls Mean (SD)* FII 98 (35) 111 (18) FVII 91 (35) 112 (25) FVIII 131 (44) 109 (37) VWF 138 (68) 113 (49) FIX 102 (20) 106 (19) FX 100 (42) 117 (20) FXI 100 (19) 101 (20) Fibrinogen 4.0(0.8) 3.0(0.6) Blood group (%) (%) O Non-O Factor II, VII, VIII, X, and XI were expressed in IU/ml; VWF and fibrinogen in g/l; factor IX in U/dl. *SD= standard deviation Vitamin K dependent factors, figures were calculated after exclusion of patients using vitamin K antagonist treatment at time of blood draw leaving 57 patients compared with 69 patients in the analysis of the vitamin K independent factors 28

31 ), VWF (24.8 g/l, 95%CI, ), and fibrinogen (0.1 g/l, 95%CI, )). Elevated levels of fibrinogen were found in 19% of patients i.e. over 4.15 g/l. An elevated level of fibrinogen (i.e. above the 90 th percentile) was associated with a 2.9-fold (95% CI: ) increased risk of thrombosis compared with low levels (below 90 th percentile), after adjustment for age and sex. Twenty-two percent of the patients had an elevated level of factor VIII, i.e. over 155 IU/ml (table 3). Individuals with an elevated level of factor VIII had a 4.2-fold (95% CI: ) increased risk compared with those with low levels after adjustment for age and sex. Twenty percent of patients had an elevated level of VWF, i.e. over 170 g/l. Individuals with elevated levels of VWF had a 4.0-fold (95% CI: ) increased risk of venous thrombosis compared to those with low levels after adjustment for age and sex. Of all patients, 68% had blood group non-o compared with 53% of all controls. Blood group non-o was associated with a 2.1-fold (95% CI: ) increased risk of venous thrombosis compared with blood group O, after adjustment for age and sex. Associations between factor VIII, VWF and blood group non-o have been described previously 110. In control subjects, levels of factor VIII and VWF differed between blood group O and non-o. The mean difference for individuals with blood group O compared with non-o for factor VIII was 34 IU/ml (95% CI: 30-39), for VWF the mean difference was 37 g/l (95% CI: 32-41). In a model with blood group, von Willebrand factor and factor VIII levels, all effects were attenuated, particularly those of factor VIII (OR 1.7, 95%CI ( ) and Table 3. The risk of venous thrombosis of the upper extremity for procoagulant factors Procoagulant factor 90 th percentile cutt-off point N of patients >90th percentile (%) OR (95% CI) Adjusted OR (95% CI)* 3 UEDVT: effect of blood group and coagulation factor levels FII IU/ml 5(9) 1.0 ( ) 0.9 ( ) FVII IU/ml 2(4) 0.3 ( ) 0.5 ( ) FVIII IU/ml 15(22) 2.5 ( ) 4.2 ( ) VWF g/l 14(20) 2.5 ( ) 4.0 ( ) FIX 131.1U/dl 5(9) 0.9 ( ) 1.1 ( ) FX IU/ml 5(9) 0.9 ( ) 0.9 ( ) FXI IU/ml 8(12) 1.3 ( ) 1.5 ( ) Fibrinogen 4.15 g/l 13(19) 2.2 ( ) 2.9 ( ) Blood group N of patients blood group non-o OR (95% CI) Adjusted OR (95% CI)* Non O vs. O - 47(68) 1.9 ( ) 2.1 ( ) *Adjusted for age and sex Vitamin K dependent factors, figures were calculated after exclusion of patients using vitamin K antagonist treatment at time of blood draw leaving 57 patients compared with 69 patients in the analysis of the vitamin K independent factors 29

32 3 UEDVT: effect of blood group and coagulation factor levels VWF (OR 1.9, 95%CI ( )), while blood group non-o remained associated with an 1.7-fold risk (95% CI: ). None of the patients had a protein S deficiency. Only one patient had low protein C levels and only one patient low antithrombin levels. Therefore, the thrombotic risk for these anticoagulant factors could not be estimated. Increased risks of venous thrombosis were seen for elevated levels of factor VIII, VWF, and fibrinogen. To explore a dose response relation between the levels of these factors and the risk of venous thrombosis, factor levels were divided in tertiles. Cut-off values for tertiles (in the controls) and risk estimates are shown in table 4. With the lowest tertile as a reference category, there was a clear dose response relation for both VWF and factor VIII levels. For fibrinogen levels both the second and third tertile showed similarly increased risks. Since many female patients were using oral contraceptives at time of the thrombotic event and previous studies showed a high joint effect of oral contraceptive use and elevated factor VIII levels in patients with a venous thrombosis of the leg, we considered the possibility of a synergistic effect between elevated levels of factor VIII and oral contraceptive use. Results are shown in table 5. An elevated level factor VIII alone as well as use of oral contraceptive use only, were associated with an increased risk of venous thrombosis of the upper extremity (OR 5.3, 95%CI ( ) and 7.5, 95%CI ( ) respectively). The thrombotic risk was highest for women with both elevated levels of factor VIII and oral contraceptive use (OR 30.5, 95%CI ( )). Table 4. Factor VIII, VWF, and fibrinogen and the risk of venous thrombosis of the upper extremity Coagulation factor (cut-off points for tertiles) 1 st Tertile 2 nd Tertile* 3 rd Tertile* Factor VIII (89, 120) 1# 2.2 ( ) 8.8 ( ) VWF (87, 122) 1# 2.0 ( ) 5.9 ( ) Fibrinogen (3.0, 3.5) 1# 2.5 ( ) 2.3 ( ) * Adjusted for age and sex # Reference category Table 5. Joint effect of factor VIII and oral contraceptive use and the risk of thrombosis in the upper extremity. Oral contraceptive use High factor VIII Patients N Controls N OR (95% CI)* *Adjusted for age #Reference category # ( ) ( ) ( ) 30

33 Discussion In this study we assessed the association between levels of pro- and anticoagulant factors and blood group and the risk of venous thrombosis of the upper extremity. Elevated levels of factor VIII, VWF, fibrinogen, and blood group non-o increased the risk of a venous thrombosis of the upper extremity. Elevated levels of factor II, factor VII, factor IX, factor X, and factor XI were not associated with an increased risk. We were not able to assess the risk of venous thrombosis of the upper extremity associated with protein C, protein S, or antithrombin deficiency. 3 The overall prevalence of deficiencies of anticoagulant factors protein C, protein S, and antithrombin among patients in several studies varies between 2% up to 12% 88,89,92. In the current study no patients with protein S deficiency were identified; only one patient had a protein C deficiency, and only one patient was antithrombin deficient (both 3%). Given that the population prevalence of these abnormalities varies between 0.2% (protein C deficiency) and 0.02% (antithrombin deficiency), our findings are compatible with highly increased risks. After dichotomization at the 90 th percentile as measured in the control subjects, elevated levels of factor VIII, VWF, and fibrinogen were found to increase the risk of a first venous thrombosis of the upper extremity. There was also a clear dose response relation for factor VIII and VWF after stratification of the levels in tertiles. For fibrinogen no gradual increase in risk over the tertiles was observed. Factor VIII, VWF, and fibrinogen are acute phase proteins. Therefore the question rises whether the elevated levels are a cause or consequence of venous thrombosis of the upper extremity. Blood was drawn at least 4 months after venous thrombosis. When comparing patients with a blood draw within one year with those with a blood draw more than one year after the thrombotic event, levels of factor VIII, VWF, and fibrinogen were similar (mean differences: factor VIII (2.4 U/dl, 95%CI, ), VWF (0.9 U/dl, 95%CI, ), and fibrinogen (-0.2 g/l, 95%CI, )). Furthermore, the prevalence of reported febrile disorders at time of blood draw and mean white blood cell count were similar between patients and controls. Finally, the effect of blood group is likely to be mediated via von Willebrand factor and factor VIII levels, and of course invariant. These results indicate that it is unlikely that the elevated levels of factor VIII, VWF, and fibrinogen are due to an acute phase reaction, which corroborates what has been proposed before for patients with venous thrombosis of the leg 121. Thus, elevated levels of factor VIII, VWF, and fibrinogen are likely to be a cause of venous thrombosis of the upper extremity rather then a consequence of the disease. UEDVT: effect of blood group and coagulation factor levels Blood group non-o was associated with a two-fold increased risk of venous thrombosis of the upper extremity. After adjustment for factor VIII and VWF the risk of venous thrombosis of the upper extremity for blood group non-o compared with O was similar to the crude risk estimate, indicating that blood group non-o increases the risk of venous thrombosis via a 31

34 3 different pathway than factor VIII and VWF levels. After additional adjustment for bloodgroup and VWF or factor VIII, the risk of venous thrombosis associated with elevated factor VIII levels decreased from a 4.2 to a 1.7-fold increased risk, whereas the risk associated with elevated VWF levels decreased from a 4.0 to a 1.9-fold increased risk. This shows that the effect of factor VIII and VWF is partly due to the effects of blood group non-o. There was a synergistic effect seen for high levels of factor VIII and oral contraceptive use, which was supra-additive. UEDVT: effect of blood group and coagulation factor levels This is the first study that assessed the risk of venous thrombosis of the upper extremity associated with elevated procoagulant factors. Although the number of patients was low due to the exclusion of individuals with a malignancy, we found an increased risk of upper extremity thrombosis for patients with an elevated level of factor VIII, VWF, and fibrinogen. The results for VIII, VWF, fibrinogen, and blood group non-o were similar to results found for venous thrombosis of the leg. Due to the small number of patients with anticoagulant protein C, protein S, and antithrombin deficiency, we were unable to estimate the risk of venous thrombosis of the upper extremity associated with low levels of anticoagulant factors, although the finding of two patients among 69 with such a defect is highly suggestive of an increased risk. This study showed that elevated levels of several of the procoagulant factors are risk factors for both deep venous thrombosis of the leg as well as venous thrombosis of the upper extremity. However, there are also differences with regard to coagulation factors as risk factor for a venous thrombosis of the leg or of the upper extremity. Factors II, IX and XI have been described as risk factors for venous thrombosis of the leg but do not appear to increase the risk of venous thrombosis of the upper extremity in our study. This might be explained by the number of patients in our study. Although our study is the largest study investigating the effect of coagulation factors on venous thrombosis in the upper extremity among individuals without a malignancy, we only included 69 patients. A hypothetical explanation for the different coagulation factors that give an increased risk for deep venous thrombosis of the leg and or the upper extremity could be that venous thrombosis of the leg is likely to be more often induced by stasis (plaster cast, travel, hospital admission) and venous thrombosis of the upper extremity more often by injury of the vessel wall (CVCs, physical effort). In the presence of an injury of the vessel wall it might be important that factors related to the acute phase are elevated compared to other factors. If venous thrombosis is due to stasis, it might be less important which procoagulant abnormality is present. However, this hypothesis should be tested in a different study. In conclusion, we found an increased risk of venous thrombosis of the upper extremity in patients with an elevated level of fibrinogen, factor VIII, VWF, and in patients with blood group non-o. However, due to the low number of patients the confidence intervals are wide. 32